Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is a progressive, fatal neurodegenerative disease that is characterized by degeneration of motor neurons in the cortex, brainstem, and spinal cord, leading to paralysis and eventual death. In approximately 10 percent of ALS cases, the disease is inherited; approximately one-fifth of these familial cases are associated with mutations in sod1, the gene that encodes the human antioxidant enzyme copper-zinc superoxide dismutase (CuZnSOD). These mutations are the only known cause of ALS and therefore represent the strongest clues available in investigating what causes ALS, whether inherited or sporadic. Over 90 individual mutations in CuZnSOD are known to cause ALS, and it is widely accepted that they exert their toxic effects by a gain of function mechanism. Several plausible mechanisms have been suggested to explain the nature of the toxic function gained by CuZnSOD as a consequence of the ALS mutations. The proposed mechanisms include a new chemical reactivity for CuZnSOD, formation of CuZnSOD aggregates, co-aggregation of other unknown components with CuZnSOD, and disruption of metal ion homeostasis due to binding or release from CuZnSOD. Each of these mechanisms would appear to be dependent on a change in structure or on a change in conformational stability or flexibility of the mutant protein relative to the extremely stable wild-type protein. It is therefore important to determine the physical properties of the mutant enzymes, particularly with respect to structure, stability, dynamics, and metal binding and to assay new and potentially toxic chemical reactivities. The primary objective of this project is to discover how mutations in CuZnSOD cause ALS and to explore the possibility that CuZnSOD is also linked to sporadic, i.e., non inherited forms, of ALS. The specific aims are as follows: to expand numbers of isolated, properly metallated and N-acetylated ALS-mutant CuZnSOD proteins for structural and mechanistic characterization, to determine the thermal stabilities and thermodynamics of metal ion binding of wild type and ALS-mutant CuZnSOD, to compare the dynamic properties of wild type and ALS-mutant CuZnSOD, to isolate and characterize mixed dimers consisting of one subunit each of ALS-mutant and wild type CuZnSOD (analogous to the mixed dimers that are expected to occur in ALS patients), to prepare chemically modified wild type CuZnSODs for comparison of properties with ALS-mutant CuZnSOD as possible models for sporadic ALS, to study reactions of ALS-mutant CuZnSOD with ascorbate and with hydrogen peroxide, to continue whole cell studies of yeast strains expressing wild type and ALS mutant CuZnSODs as their only CuZnSODs, and to develop in vivo inhibitors of wild type and ALS-mutant CuZnSODs.